This invention relates to methods and compositions for detecting N-glycosylase activity. More particularly, this invention relates to sensitive detection of N-glycosylase toxins based on their biological activity.
Ribosome-inactivating proteins (RIPs) include adenine-specific N-glycosylases, such as ricin, saporin, and gelonin, which depurinate ribosomal RNA to cause irreversible inhibition of protein synthesis. L. Barbieri et al., “Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites,” 286 Biochem. J. 1–4 (1992); M. Zamboni et al., “High-pressure-liquid chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin,” 259 Biochem. J. 639–643 (1989). Alternatively referred to as (adenosine) N-glycosidases when acting on RNA, these enzymes also remove adenine from DNA molecules, including single-stranded or denatured DNA. E. Nicolas et al., “Gelonin is an unusual DNA glycosylase that removes adenine from single-stranded DNA, normal base pairs and mismatches,” 275 J. Biol. Chem. 31399–31406 (2000); L. Barbieri et al., “Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A),” 25 Nucleic Acids Res. 518–522 (1997). Likewise, uracil DNA glycosylase (UDG) can remove uracil from deoxyuridine residues in oligodeoxyribonucleotides (ODNs). N. V. Kumar & U. Varshney, “Contrasting effects of single stranded DNA binding protein on the activity of uracil DNA glycosylase from Escherichia coli towards different DNA substrates,” 25 Nucleic Acids Res. 2336–2343 (1997). The resulting abasic sites have intact phosphodiester backbones when purified N-glycosylases are used, L. Barbieri et al., “Polynucleotide:Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA,” 128 J. Biochem. (Tokyo) 883–889 (2000), although backbone cleavage (lyase activity) has been reported. E. Nicolas et al., “A new class of DNA glycosylase/apurinic/apyrimidinic lysases that act on specific adenines in single-stranded DNA,” 273 J. Biol. Chem. 17216–17220 (1998).
N-Glycosylases, when naturally coupled to cell-binding lectins, are also known as potential bioterrorism agents because of their toxic properties. These toxins remove adenine residues from ribosomal RNA, thereby inactivating ribosomes and inhibiting protein synthesis, which are required for viability of cells. Ricin and abrin are two biothreats that contain N-glycosylases.
Activity-based assays previously reported for N-glycosylases involve radiolabeled DNA substrates, M. Brigotti et al., “A rapid and sensitive method to measure the enzymatic activity of ribosome-inactivating proteins,” 26 Nucleic Acids Res. 4306–4307 (1998); L. Xia & T. R. O'Connor, “DNA glycosylase activity assay based on streptavidin paramagnetic bead substrate capture,” 298 Anal. Biochem. 322–326 (2001), or the derivatization of released adenine to a fluorescent product using chloroacetaldehyde, which is highly toxic. M. Zamboni et al., supra; W. H. Lawrence et al., “Toxicity profile of chloroacetaldehyde,” 61 J. Pharm. Sci 19–25 (1972). In both cases, separation of unreacted DNA substrate was required. The classic approach for assaying RIPs uses inhibition of in vitro translation as a measure of RIP activity. Typically, the complex, unstable reagents necessary for this approach are derived from lysates of rabbit reticulocytes. M. Langer et al., “A nonradioactive assay for ribosome-inactivating proteins,” 243 Anal. Biochem. 150–153 (1997). Since ribosomal inactivation produces a signal that is inversely proportional to the amount of RIP present, inhibitory substances other than RIPs may cause false responses. A fluorescence-based assay has been reported, which directly measures N-glycosylase activity on short ODN substrates having defined sites for enzyme action, but this method is not conducive to signal amplification and thus is limited in sensitivity. E. L. Kreklau et al., “A novel fluorometric oligonucleotide assay to measure O(6)-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase,” 29 Nucleic Acids Res. 2558–2566 (2001).
Thus, while prior N-glycosylase assays are known and are generally suitable for their limited purposes, they possess certain inherent deficiencies that detract from their overall utility in field-testing for bioterrorism agents.
In view of the foregoing, it will be appreciated that providing a potentially field-deployable assay for detecting N-glycosylase activity would be a significant advancement in the art.